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. 2018 Jul 6;46(12):6140-6151.
doi: 10.1093/nar/gky457.

Origin Recognition Is the Predominant Role for DnaA-ATP in Initiation of Chromosome Replication

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Free PMC article

Origin Recognition Is the Predominant Role for DnaA-ATP in Initiation of Chromosome Replication

Julia E Grimwade et al. Nucleic Acids Res. .
Free PMC article

Abstract

In all cells, initiation of chromosome replication depends on the activity of AAA+ initiator proteins that form complexes with replication origin DNA. In bacteria, the conserved, adenosine triphosphate (ATP)-regulated initiator protein, DnaA, forms a complex with the origin, oriC, that mediates DNA strand separation and recruitment of replication machinery. Complex assembly and origin activation requires DnaA-ATP, which differs from DnaA-ADP in its ability to cooperatively bind specific low affinity sites and also to oligomerize into helical filaments. The degree to which each of these activities contributes to the DnaA-ATP requirement for initiation is not known. In this study, we compared the DnaA-ATP dependence of initiation from wild-type Escherichia coli oriC and a synthetic origin (oriCallADP), whose multiple low affinity DnaA sites bind DnaA-ATP and DnaA-ADP similarly. OriCallADP was fully occupied and unwound by DnaA-ADP in vitro, and, in vivo, oriCallADP suppressed lethality of DnaA mutants defective in ATP binding and ATP-specific oligomerization. However, loss of preferential DnaA-ATP binding caused over-initiation and increased sensitivity to replicative stress. The findings indicate both DnaA-ATP and DnaA-ADP can perform most of the mechanical functions needed for origin activation, and suggest that a key reason for ATP-regulation of DnaA is to control replication initiation frequency.

Figures

Figure 1.
Figure 1.
Characterization of low affinity DnaA binding sites in Escherichia coli oriC. (A) Map of oriC, showing low affinity DnaA recognition sites that preferentially bind DnaA-ATP (gray boxes) and high and low affinity sites that bind both nucleotide forms (black and hatched boxes, respectively). The putative τ1 site is marked with a dashed outline. The DUE is indicated. (B) DMS footprint comparing in vitro binding of purified DnaA-ATP and DnaA-ADP to oriC. Binding sites and positions of the G2 and G4 residues diagnostic of DnaA binding are marked. (C) Quantitation of G2 or G4 band intensities for each site when bound to 80 nM DnaA-ATP (gray) or DnaA-ADP (black), relative to band intensity of site in the absence of DnaA.
Figure 2.
Figure 2.
DnaA-ATP oligomerization is not required for cooperative binding between low affinity sites. (A) EMSA of wild-type DnaA-ATP, DnaA(R285A)-ATP or DnaA-ADP binding to probe carrying the closely spaced sites shown below the panels. DnaA:DNA molar ratios were 0:1, 2.5:1 and 10:1. Positions of unbound probe (0), and probe bound to 1, 2 or 3 molecules of DnaA are marked. (B) DMS footprints of DnaA-ATP, DnaA-ADP and DnaA(R285A)-ATP bound to oriCallADP. Binding sites and diagnostic G2 and G4 residues are marked. (C) Quantitation of G2 or G4 band intensities for each site when bound to 80 nM DnaA-ATP (gray), DnaA-ADP (black) or DnaA(R285A)-ATP (stippled) relative to intensities at 0 nM DnaA.
Figure 3.
Figure 3.
DnaA-ADP is active in unwinding oriCallADP, but not wild-type oriC. Purified supercoiled plasmid was incubated with the indicated concentrations of DnaA-ATP (A), or a mixture of DnaA-ATP and DnaA-ADP at the concentrations indicated in (B), and treated with P1 nuclease. Topological forms were separated by electrophoresis through agarose gels, followed by staining with ethidium bromide. The positions of uncut supercoiled, nicked and linearized forms are shown. The percentage of DNA converted to linear form was quantified.
Figure 4.
Figure 4.
Initiation from oriCallADP has a reduced requirement for DnaA-ATP in vivo. (A) Cells from a single colony of MG1655(dnaA46 ts)(bottom) or MG1655(oriCallADP, dnaA46 ts) (top) were diluted, plated and incubated at either 25°C (left) or 42°C (right). Colonies were counted after 24 or 18 h, respectively. Representative plates are shown. (B) Quantitation of the plating study shown in (A). Colony counts from two separate experiments, with triplicate plates for each temperature in each experiment were run. Error bars are SD of mean. (C) DnaA null cells, carrying both a plasmid R1 origin and either oriC (gray bars) or oriCallADP (black bars), were transformed with a plasmid expressing the plasmid R1 origin inhibitor copA or a plasmid co-expressing either wild-type DnaA or DnaA(R285A) and copA. Transformation efficiency was calculated after 18 h of growth. Two separate experiments, with triplicate plates were run. Error bars are SD of mean. (D) Oligomeric DnaA forms after DTSSP treatment of purified supercoiled plasmids incubated with 0 or 100 nM DnaA-ATP or DnaA(R285)-ATP. Three separate experiments were performed. Quantitation is shown in Supplementary Figure S3.
Figure 5.
Figure 5.
Over-initiation in oriCallADP cells leads to increased sensitivity to replicative stress. (A and B) DNA histograms of wild-type oriC (A) or oriCallADP (B) cells growing in LB (top panels), or minimal media supplemented with glucose, casamino acids and uracil (bottom panels), treated with cephalexin and rifampicin, and analyzed by flow cytometry. The number of origins in the cells at the time of drug treatment is shown. (C) Survival fraction of cells incubated in the presence of AZT or HU are plotted after 18 h of growth. Two separate experiments, with triplicate plates were run. Error bars are SD of mean.

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